The present study aimed to environmentally friendly synthesis of ZnO NPs using Fioria vitifolia leaf extracts which provides a sustainable and green approach for production of NPs. The produced ZnO NPs were evaluated using various spectrum approaches (UV-vis, FTIR XRD, TEM and EDAX). The synthesized ZnO NPs was confirmed by UV-Visible spectroscopy exhibited a peak at 370 nm. SEM imaging revealed a flash-like and needle-like bottom morphology. Fourier-transform infrared spectroscopy (FTIR) analysis detected vibrations corresponding to alcohols, halides, and aromatics functional groups. TEM showed spherical-shaped NPs with an average diameter of 11 nm. XRD analysis exhibited distinct peaks at 2θ values of 31.7°, 34.3°, 36.2°, 47.4°, 56.6°, 62.8°, 66.4°, 67.9°, 69.1°, and 76.8°, corresponding to the crystallographic planes (100), (002), (101), (102), (110), (103), (200), (112), (201), (004), and (202) planes respectively. The antibacterial activity demonstrated significant zones of inhibition against E. coli (17 ± 0.6 mm) and S. aureus (23.7 ± 0.5 mm), and inhibition of biofilm formation in S. aureus and C. albicans. Additionally, S. mutans exhibited the highest sensitivity to the minimum inhibitory concentration (MIC) of ZnO NPs, with complete inhibition occurring at 7.5 μg/mL. Furthermore, antioxidant DPPH assays exhibited IC values of 42 μg/mL. Additionally, the anti-inflammatory properties of ZnO NPs of F. vitifolia were evaluated in-vitro using models utilizing the human red blood cells (HRBC) membrane stabilization method (MSM), and it was shown to have an MSM of 83.87 % at 250 μg/mL. Furthermore, ZnO NPs exhibited anticancer activity against the MDA-MB-231 breast cancer cell line with an IC value of 35.50 μg/mL. Toxicological evaluation of FV-ZnO nanoparticles in zebrafish (Danio rerio) embryos indicated low toxicity at maximum concentration. These is first findings suggest that ZnO NPs synthesized from F. vitifolia leaf extracts possess significant antibacterial, antioxidant, anti-inflammatory, and anticancer properties. Additionally, their low toxicity in zebrafish embryos makes them suitable for further development in antimicrobial therapies with minimal side effects, offering a sustainable, biocompatible solution to tackle multidrug-resistant microbial infections.